Reagents and methods for detecting influenza

ABSTRACT

Two universally conserved sequences from influenza type A neuraminidases were identified by large scale sequence analysis then chemically modified and conjugated to carrier proteins to generate mono-specific and monoclonal antibodies. The two antibodies, one targeting the N-terminus of the type A neuraminidase and the other sequence close to enzymatic active site, were capable of binding to all 9 subtypes of neuraminidase while demonstrating remarkable specificity against the viral neuraminidase sequences since no cross-reactivity against allantoic proteins was observed. Quantitative analyses of NA using slot blot suggest that the antibodies can be used for NA antigen quantitation in vaccines. These represent the first time the antibody-based immunoassay can be used for NA quantitative determination.

This application is a divisional application of U.S. Ser. No.14/556,415, filed Dec. 1, 2014 which was a divisional application ofU.S. Ser. No. 13/322,459, filed Nov. 24, 2011 which was a 371 of PCTApplication CA10/00784, filed May 28, 2010, now abandoned, which claimedthe benefit of U.S. Provisional Patent Application U.S. Ser. No.61/182,920 filed on Jun. 1, 2009.

FIELD OF THE INVENTION

The present invention relates to reagents and methods for detectinginfluenza virus proteins, in particular neuraminidase (NA). Inparticular, the present invention relates to peptide conjugates,antibodies, and use of antibodies for detecting neuraminidase in asample, especially for verifying potency of influenza vaccines.

BACKGROUND OF THE INVENTION

Influenza can infect as much as 5-15% of the world population, resultingin 3-5 million cases of severe illness and up to 500,000 deaths eachyear. In the U.S. alone, flu epidemics lead to approximately 300,000influenza-related hospital admissions and 36,000 influenza-relateddeaths annually in addition to an estimated cost of $12 billion per year(Poland 2001; Simonsen et al. 2007, PMID: 17897608). Current seasonalinfluenza vaccines are produced with strains recommended by the WorldHealth Organization about 9-12 months ahead of the targeted season(Carrat et al. 2007). The vaccines typically contain two type Ainfluenza strains and one type B strain, which are predicted to be themost likely strains to cause the upcoming flu epidemic.

However, there are inherent disadvantages associated with thepreparation of conventional influenza vaccines such as the uncertaintyof the actual circulating strain, the need for annual updating of themanufacturing process and preparation of reagents for vaccine lotrelease. Furthermore, mismatches between the strains selected forvaccine preparation and the circulating viruses were found to beresponsible for much reduced efficacy of the seasonal influenza vaccines(Bridges et al. 2000; De Filette et al. 2005). Clearly, the drawbacksassociated with traditional vaccine preparation would be drasticallyexacerbated in the event of an outbreak of pandemic influenza, given aperceivably much shortened timeframe available for the production ofprophylactic vaccines for global needs. All these problems concerningthe influenza vaccines are largely due to one single biological propertyof the influenza virus itself, i.e. the constant mutations of the virussurface proteins hemagglutinin (HA) and neuraminidase (NA).

Currently, influenza A viruses representing 16 HA and 9 NA subtypes havebeen detected in wild birds and poultry throughout the world (Zambon1999; Treanor 2004; Fouchier 2005). Frequent antigenic drifting orshifting of HA and NA prompted numerous exploratory investigations ofvaccines that are intended to induce host immune responses against viralproteins that are less subject to antigenic fluctuations. Of theseconserved antigenic determinants, the nucleoproteins (NP) and Matrix (M)have been shown to induce protective immunity against diverse strains ofthe viruses (Frace et al. 1999; Epstein et al. 2002; de Filette 2005;Mozdzanowska et al. 2003; Fan et al. 2004).

Furthermore, it has been suggested that cell-mediated immune responserather than humoral immune responses protect the animals immunized withNP-based vaccines while antibody-mediated protections against lethalchallenges of various subtypes of influenza virus were reported with theuse of M2-based vaccines (Neirynck et al. 1999; de Filette et al 2005;Mozdzanowska et al. 2003). None of these universal vaccines appears toprevent viral infection in animal studies although prevention ofclinical diseases was found to be promising (Gerhard et al. 2006).

Given the importance of neutralizing antibodies against NA in preventinginfluenza infection, the conserved regions in the NA proteins have alsoreceived great attention in recent years.

There remains a need in the art for reagents that may be universallyused to detect influenza viruses or proteins therein, especiallyneuraminidase (NA) proteins.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided acomposition comprising an influenza neuramidase peptide consisting ofthe amino acid sequence as set forth in SEQ ID No. 1 or SEQ ID No. 2attached to a first end of a spacer, said spacer being attached at asecond end thereof to a carrier protein.

According to a second aspect of the invention, there is provided acomposition comprising an influenza neuramidase peptide consisting ofthe amino acid sequence as set forth in SEQ ID No. 1 or SEQ ID No. 2attached to a first end of a spacer, said spacer being attached at asecond end thereof to a first end of a linker, said linker beingattached at a second end thereof to a carrier protein.

According to a third aspect of the invention, there is provided a methodof inducing an immune response in an individual in need of or desirousof such treatment comprising administering to said individual aneffective amount of the composition as described above.

According to a fourth aspect of the invention, there is provided amethod of preparing antibodies against influenza virus neuraminidasecomprising inoculating an animal with an effective amount of thecomposition as described above and after the animal has producedantibodies against said composition, recovering said antibodies fromsaid animal.

According to a fifth aspect of the invention, there is provided a methodof preparing monoclonal antibodies against influenza virus neuraminidasecomprising inoculating an animal with an effective amount of thecomposition as described above, removing antibody-producing cells fromsaid animal, fusing a respective one of said antibody-producing cellswith a respective one of an immortal cell line, thereby producing arespective one hybridoma cell, and selecting for hybridoma cellsproducing antibodies against said composition.

According to a sixth aspect of the invention, there is provided a methodof determining the potency of an influenza vaccine preparationcomprising providing an influenza vaccine preparation to be tested anddetermining the amount of neuraminidase in said vaccine preparationusing antibodies prepared according to one of the methods describedabove, wherein higher levels of neuraminidase are indicative of a morepotent vaccine preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts sequence similarity of influenza A virus neuraminidases.A total of 8813 full length, non redundant influenza type A NA sequencesrepresenting any host or HXNX subtype were downloaded from the NCBIinfluenza Virus resource database, including Pandemic (H1N1) 2009viruses, and Flu Project Sequences, Shannon entropy was calculated foreach position of amino acid of the identified consensus sequences todetermine the degree of variation. FIG. 1 represents the highlyconserved epitope designated HCA-2, near the enzymatically active siteof all NA enzymes. For the NA of /A/California/07/2009, the epitope islocated at amino acid positions from 221-230 (N2 numbering).

FIG. 2 depicts sequence similarity of influenza A virus neuraminidases.A total of 8813 full length, non redundant influenza type A NA sequencesrepresenting any host or HXNX subtype were downloaded from the NCBIinfluenza Virus resource database, including Pandemic (H1N1) 2009viruses, and Flu Project Sequences, Shannon entropy was calculated foreach position of amino acid of the identified consensus sequences todetermine the degree of variation. FIG. 2 represents the universallyconserved epitope designated HCA-3, at the N-terminus of all NA type Aenzymes.

FIG. 3 depicts binding of Mono-specific and monoclonal antibodies to theHCA-2 and HCA-3 epitopes in direct ELISA. HCA-2 or HCA-3 free peptides,GST-peptide or recombinant NA (rNA) were coated, respectively on 96-wellplates, following by reaction with the antibodies and 2^(nd) goatanti-rabbit IgG peroxidise conjugates. The left side of the figurerepresents the reactions with non-specific polyclonal antibodies andtheir pre-bleed controls while the right side of the figure describesthe reactions with the monoclonal antibodies (MAbs) derived from thehybridoma supernatants and their culture media controls. The data showsthat HCA-2 or HCA-3 mono-specific antibodies (1:4000 dilution) or MAbs(undiluted hybridoma supernatants) bind to their respective epitopes inthe free peptides, GST-peptides or rNA. Same results were obtained fromaffinity-purified antibodies.

FIG. 4. Quantitative detection of NA using antibodies against NA. The NAantigens were serially diluted in PBS-containing 0.01% Zwittergent(final concentration) and blotted onto PVDF membrane. The membrane isthen incubated with the universal antibodies HCA-2 Monoclonalantibodies, followed by detection with anti-rabbit IgG peroxidaseconjugate. Slot blot was conducted as described (Li C. et al. 2010).

FIG. 5. Standard curve for the quantification of NA by HCA-2. Thecurrently accepted 4-parameter logistic (4-PL) model was employed forthe calibration curve fitting in the immunoassays as described (Chun etal 2008) showing that there is a relationship between the amount ofsignal and the amount of protein. Therefore, antibodies to HCA-2 can beused as a quantitative detection method for detection of NA.

FIG. 6. Quantitative detection of NA using antibodies against NA. The NAantigens were serially diluted in PBS-containing 0.01% Zwittergent(final concentration) and blotted onto PVDF membrane. The membrane isthen incubated with the HCA-3 monoclonal antibodies, followed bydetection with anti-rabbit IgG peroxidase conjugate. Slot blot wasconducted as described (Li C. et al. 2010).

FIG. 7. Binding of HCA-2 and HCA-3 antibodies to 9 subtypes of NAproteins. Allantoic fluids of 9 NA subtypes of influenza virusespropagated in embyronated eggs were fractionated by SDS-PAGE, followedby detection of the NA proteins using the HCA-2 (upper panel) and HCA-3antibodies (middle panel). Rabbit polyclonal anti-NP proteins ofinfluenza viruses were used as another control (lower gel panel). “+”represents allantotic fluids spiked with rNA or A/New Caledonia/20/99reacting with the corresponding anti-NA antisera as positive controlwhile the negative controls (−) were allantoic fluids from un-infectedeggs.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference.

The neuraminidases of influenza type viruses play important roles inviral replication by facilitating the release of viral particles fromthe infected cells. Neuraminidase specific-antibodies alone have beenreported to protect animals from lethal challenge and therefore are animportant component of an effective influenza vaccine. However, theamount of neuraminidase in current vaccines is typically not determinedbecause of a lack of appropriate reagents, methods and internationalreferences standards. Thus, a simple and accurate method capable ofquantifying neuraminidase would be useful for better quality control ofinfluenza vaccines.

Antigenic drifting and shifting of the influenza A viruses havepresented to the scientific community a daunting challenge in terms ofepidemiological monitoring, vaccine development and quality control. Wehave now generated and characterized antibodies against the mostconserved region in the neuraminidase of influenza A viruses.Bioinformatics analyses of all available neuraminidase (NA) sequencesfrom public domain revealed two stretches of amino acids comprised ofILRTQES(E/S)C (SEQ ID NO: 1) which is found at amino acids 223-231 andMNPNQKIITIGS (SEQ ID NO: 2) which is found at the N-terminus of NA. Aswill be appreciated by one of skill in the art, the peptide as set forthis SEQ ID No. 1 can also be expressed as ILRTQESEC (SEQ ID NO: 3) andILRTQESSC(SEQ ID NO: 4). These regions were found to be present in allviral strains with minor substitutions.

As discussed herein, in one aspect of the invention, there is provided acomposition comprising an influenza neuramidase peptide consisting ofthe amino acid sequence as set forth in SEQ ID No. 1 or SEQ ID No. 2attached to a first end of a spacer, said spacer being attached at asecond end thereof to a carrier protein. Preferably, the neuramidasepeptide is attached at its C terminus to the spacer.

As will be appreciated by one of skill in the art, any suitable spacermay be used, for example, but by no means limited to amino acids,peptides, phosphoramidite, ε-aminohexanoic acid and 6-aminocaproic acid.In a preferred embodiment, the spacer is 6-aminocaproic acid.

The carrier protein may be any suitable carrier protein known in theart. As will be well known to those of skill in the art such carrierproteins are routinely used for ‘presenting’ antigens and/or epitopesfor eliciting an immune response. As such, the selection of carrierprotein depends largely on the intended use and is well within routineskill in the art and does not require undue experimentation. Forexample, the carrier protein may be selected from the group consistingof keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), rabbitserum albumin (RSA), ovalbumin (OVA), thyroglobulin (THY) and humangamma globulin (HGG).

As discussed herein, there is also provided a composition comprising aninfluenza neuramidase peptide consisting of the amino acid sequence asset forth in SEQ ID No. 1 or SEQ ID No. 2 attached to a first end of aspacer, said spacer being attached at a second end thereof to a firstend of a linker, said linker being attached at a second end thereof to acarrier protein.

As will be appreciated by one of skill in the art, any suitable linkermay be used. In some embodiments, the linker is selected from the groupconsisting of an amino acid, a peptide of 2-10 amino acids and KKC.

In a preferred embodiment, the linker is KKC and the spacer is6-aminocaproic acid.

The compositions described herein can also be used to elicit an immuneresponse, for example, as a vaccine, or for the harvesting ofantibodies.

For example, in some embodiments, there is provided a method of inducingan immune response in an individual in need of or desirous of suchtreatment comprising administering to said individual an effectiveamount of the composition. As will be appreciated by one of skill in theart, ‘an effective amount’ in this context refers to an amount that issufficient to induce the desired immune response. It is of note that theeffective amount may be administered in one or more doses, for example,as a first initial inoculation followed by one or several additional‘booster’ inoculation(s).

In other embodiments, there is provided a method of preparing antibodiesagainst influenza virus neuraminidase comprising inoculating an animalwith an effective amount of the composition as described above and afterthe animal has produced antibodies against said composition, recoveringsaid antibodies from said animal.

In further embodiments, there is provided a method of preparingmonoclonal antibodies against influenza virus neuraminidase comprisinginoculating an animal with an effective amount of the composition asdescribed above, removing antibody-producing cells from said animal,fusing a respective one of said antibody-producing cells with arespective one of an immortal cell line, thereby producing a respectiveone hybridoma cell, and selecting for hybridoma cells producingantibodies against said composition.

In other aspects of the invention, there are provided antibodiesprepared according to the above methods. As discussed herein, theseantibodies may be used in the preparation of a medicament orpharmaceutical composition or as reagents for detecting neuraminidase,as discussed below. As discussed herein and as will be readily apparentto one of skill in the art, such antibodies may be purified or otherwiseisolated first using means known in the art.

As will be appreciated by one of skill in the art and as discussedherein, these antibodies may be used for a variety of purposes.

For example, in some embodiments, there is provided a method ofdetermining the potency of an influenza vaccine preparation comprisingproviding an influenza vaccine preparation to be tested and determiningthe amount of neuraminidase in said vaccine preparation using antibodiesprepared according to one of the methods described above, wherein higherlevels of neuraminidase are indicative of a more potent vaccinepreparation. As will be appreciated by one of skill in the art and asdiscussed below, higher levels of neuraminidase are indicative of a morepotent vaccine. As such there is not necessarily a ‘threshold’ levelwhich must be attained but rather it is clear that vaccine preparationscontaining higher levels of neuraminidase will be more effective thanpreparations containing lower levers of neuraminidase.

To overcome the weak immunogenicity and insolubility of the peptides, welinked the peptides to 6-aminocaproic acid, followed by the addition ofa charged tripeptide (KKC) before its conjugation to the carrier proteinKLH (keyhole limpet hemocyanin). We found rabbits generated specificantibodies which recognized NA. The 6-aminocaproic acid spacer wasimportant in inducing antibodies against the peptides.

As discussed below, the specificity of the antibodies were assayed inWestern Blot using a wide range of subtypes of influenza A viruses(N1-N9) in crude allantoic fluid preparations. The antibodies were foundto bind all NAs with similar intensities while demonstrating nocross-reactivities to egg proteins. Moreover, versatility of theantibodies was demonstrated in a variety of immunoassays, making themuseful reagents for quantitative analyses of seasonal and candidatepandemic influenza vaccines as well as useful research tools inlaboratory settings. To our knowledge, this is the first report on thegeneration of antibodies exclusively against the peptide region whichcan potentially recognize all strains of neuraminidases. Theavailability of the antibodies enables vaccine developers to quantifythe neuaminidase in the vaccine preparations.

Any suitable spacer molecule or combination of spacer molecules may beused. Spacer molecules include, for example, amino acids, peptides,phosphoramidite, ε-aminohexanoic acid and 6-aminocaproic acid. In someembodiments, more than one spacer molecule may be used, but care must betaken to avoid solubility problems because some spacers such as6-aminocaproic acid itself are hydrophobic. The spacer moleculepreferably comprises 6-aminocaproic acid.

The carrier protein may be any suitable carrier, for example keyholelimpet hemocyanin (KLH), bovine serum albumin (BSA), rabbit serumalbumin (RSA), ovalbumin (OVA), thyroglobulin (THY) and human gammaglobulin (HGG). As will be appreciated by one of skill in the art, suchcarrier proteins are routinely used interchangeably by those of skill inthe art in different systems for presenting antigens and/or epitopes foreliciting an immune response. It is noted that substitution of onecarrier protein for another, depending on the intended use, is wellwithin ordinary skill in the art and would not require undueexperimentation.

In some embodiments, the conserved influenza virus peptide modified withthe spacer may be further modified with an appropriate linker. Thelinker is preferably an amino acid or a peptide having 2-10 amino acids.The linker may facilitate linking the modified peptide to the carrierprotein. Generally, the linker links the peptide to the carrier protein.The linker may be chosen to provide further useful properties, forexample, to facilitate dissolution of the modified peptide in aqueoussolution or to better expose the epitope for antibody generation. Insome embodiments, a tripeptide linker, particularly KKC, is preferred.

To raise antibodies against the conserved influenza virus peptide, amammal may be inoculated with a conjugate of the present invention. Someexamples of mammals are rabbit, mouse, rat, hamster, human, deer. Theantibodies so raised are preferably purified for further use.

The antibodies may be used for detecting and/or quantifying the presenceof influenza neuraminidase (NA) proteins in a sample. The antibodies areuseful for detection and/or quantification of NA proteins from manydifferent influenza virus strains, including influenza A and B strains.The universality of the antibodies makes them excellent reagents fordetermining the potency of influenza vaccines. The antibodies may alsobe used in earlier stages of seasonal influenza vaccine manufacturing,for example, to estimate presence and/or potency of influenza NAproteins prior to availability of the subtype specific antisera. Thesewould shorten the production of the vaccine and better control theneuraminidase in the manufacturing process. Specifically, neuraminidasecan be ‘lost’ during lengthy manufacturing processes, and the antibodiesdescribed herein or made by the methods described herein would allowvaccine manufacturers to know where and how the neuraminidase is lost.The antibodies also permit manufacturing of seasonal influenza vaccineat least 2-3 months ahead of the current schedule, thus greatlyfacilitating prompt production of the seasonal influenza vaccines andtimely release of the vaccines prior to an upcoming flu season.

Numerous attempts have been made in the past by various groups togenerate antibodies against the most conserved regions in the NAproteins. Two regions have been identified to be the most conservedregions among all subtypes of influenza A (Jackson et al. 1991; Gerhardet al. 2006). There have been no reports of antibodies that are specificagainst the highly conserved peptide regions only. Generation of theantibodies against the conserved regions is important because it enableshealth departments to be well ahead in pandemic flu preparedness. In theevent of a pandemic situation, it is currently impossible to havequality control (Q. C.) reagents ready for the lot-release of a pandemicflu vaccine because preparation of the reagents would take at leastseveral months and a pandemic flu could potentially spread globally in afew weeks. However, an assay system based on the present invention canalleviate this problem. Specifically, an assay system based on thepresent invention can replace the current reagents for the annual (orseasonal) flu assay as well. Currently available prior art reagents forannual flu vaccine lot release (quantitation) are prepared annually andshipped globally by the National Institute for Biological Standards andControl (NIBSC) in the U.K. or the US FDA. There are often problemsassociated with the variability of the assay among differentlaboratories and reagents are also often in short supply and cannot meetglobal needs. However, by having a universal assay system, there will beno need to prepare and ship the reagents each year globally, thussubstantially improving the quality control and simplifying theprocedure for timely lot release of seasonal (annual) flu vaccines.

By using a bioinformatics approach, we confirmed that two regions wereconserved among all subtypes of type A influenza viruses with only minorsubstitutions. Specifically, peptides most representative of theconserved peptide region were identified (HCA-2 and HCA-3). It is ofnote that both peptides were relatively hydrophobic, making it extremelydifficult to manipulate these peptides and its conjugates, i.e.,solubilization, purification, conjugation or immunoassay. In addition,previous attempts using peptides or peptide-conjugates to generateantibodies against the peptide have not been successful (Jackson et al.1999; Horvath et al. 1998), suggesting these regions are very weaklyimmunogenic.

To overcome these hurdles, we first linked the identified peptides to aspacer, 6-aminocaproic acid to improve immunogenicity, followed by theaddition of a tripeptide KKC to enable solubilization of the peptidesand conjugation of the modified peptide to the carrier protein (KLH).Through these modifications, we were successful in generating specificantibodies against the peptides. Although 6-aminocaproic acid as aspacer has been reported by others to link some haptens, e.g.,dinitrophenyl, folic acid or polysaccharide, to carrier proteins (Scottet al, 1984; Das Sarma et al. 1995; Okawa et al. 1992), the use of thespacer has not been reported for influenza peptides. At this time, itremains unclear as to how the spacer would help the host in generatingantibodies against the peptide although it is of note that two spacersmight even be better than a single one for some haptens (Scott et al.1984; Das Sarma et al. 1995; Okawa et al. 1992). While not wishing to belimited to a specific hypothesis or theory, the inventors note that itis likely that the spacer, 6-aminocarproic acid, which comprises 6carbon elements, is flexible and allows the peptide to be exposed to thesurface of the carrier proteins.

The peptides modified and conjugated in our case were found to besufficient in inducing antibodies against the immunogens(HCA-2-Acp-KKC-KLH or HCA-3-Acp-KKC-KLH). Furthermore, we found theantibodies were able to bind various recombinant NA proteins (withsimilar minor substitutions of amino acids to that of the peptides) aswell as 9 subtypes of influenza A with remarkable specificity as nocross-reactivity was observed using crude allantoic fluid preparations.

Thus, we here report two peptide regions among all influenza viruseswhich are highly conserved across all sequences of influenza strainsavailable in the public domain. However, amino acid sequences in theregion do have some minor substitutions.

We selected two peptides that are most representative of suchvariations. As discussed above, to overcome the weak immunogenicity andinsolubility of the peptides, we linked the peptides to a 6-aminocaproicacid spacer, a tripeptide (KKC) and conjugated the modified peptide toKLH. The antibodies generated in rabbits demonstrated remarkable bindingspecificities for diverse strains of influenza A viruses in ELISA andW.B. Collectively, our data indicate that the antibodies described inthis report are truly “universal” reagents for the detection of the NAproteins. Not only are they of practical application (vaccine potencytesting in the event of a pandemic flu outbreak or as replacement forthe current reagents for flu vaccine potency testing) but they are alsovaluable research tools in laboratory settings.

Further features of the invention will be described or will becomeapparent in the course of the following detailed description.

Materials and Methods: Preparation of peptides and their conjugates forimmunization.

A bioinformatics approach was employed to locate the presence of theconserved regions in the NAs. Sequences from public domains (the NCBIflu resource) were retrieved separately for each subtype. The combinedhuman and avian influenza NA sequences with identical sequences wereremoved. Next, a separate multiple alignment for each type (A and B) wasperformed, followed by the extraction of the target region from thefull-gene alignment. The Shannon entropy for each position of amino acidof the identified consensus sequences was then calculated to determinethe degree of variation. Two peptides were selected. These peptides,from influenza A, ILRTQES(E/S)C (SEQ ID NO: 1) (HCA-2); and MNPNQKIITIGS(SEQ ID NO: 2) (HCA-3) were then modified and conjugated in a proceduredescribed previously with minor modification (Wu et al. 1993; Das Samnaet al. 2005). In brief, the peptides were first linked to 6-aminocaproicacid, followed by an addition of a tripeptide (KKC). The modifiedpeptides were then conjugated to the carrier protein KLH usingsulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate(Sulfo-SMCC) as cross-linking reagent and purified according to themanufacturer's instruction manual (Fisher Canada, Nepean, ON.). Table 1summarizes the peptides (HCA-2 and HCA-3) and conjugates thereof usedfor the generation and characterization of specific antibodies againstinfluenza viruses.

Table 1 depicts the two peptides (HCA-2 and HCA-3) selected. Theselection of these peptides was based on bioinformatics analyses of allavailable influenza NA sequences and represent the most conserved aminoacid sequences in the peptide region with minor variations. HA1-C is acontrol peptide VTGLRNIPSIQSR (SEQ ID NO: 5) located at the C-terminusof NA. Acp denotes 6-aminocaproic acid, an effective spacer to linkhaptens (dinitrophenyl) to carrier proteins (Scott et al. 1984). KKCrepresent a tripeptide, which was used here to facilitate solubilizationof the carrier-free peptides in aqueous solution for antigen-antibodyinteraction in ELISA. KLH designates keyhole limpet hemocyanin.

The recombinant NAs were purchased from Proteins Sciences Corporation asdescribed previously (Wang et al. 2006). The trivalent annual influenzavaccines (1-11N1/H3N2/B) were generously provided by the NationalInstitute for the Control of Pharmaceutical and Biological products,Beijing, China.

Production of antibodies against the peptides of NAs. NZW rabbits wereobtained from Jackson Laboratory. All animal experiments were conductedin accordance with the Institutional Guidelines and Protocols for AnimalExperiments. The animals were immunized subcutaneously with varioustypes of peptide-KLH conjugate mixed with freund complete adjuvant (FCA)at 200 μg per injection, and boosted every three weeks with the samedoses of antigen in freund incomplete adjuvant.

The antibodies were purified by using the peptides as ligands inaffinity columns in a procedure described previously (Wu et al. 1993).In brief, the antisera were incubated with 5 mL of the peptide on acolumn for 10 min at room temperature, followed by washing the column atleast 5 times with PBS and 0.1% Tween™20. The antibodies were theneluted with acetate buffer (pH 2.0), followed by immediate addition ofsodium hydroxide to bring the pH to 7.2 (Wu et al., 1993).

Immunoblotting The specificities of the antibodies were determined inWestern Blot using a procedure with minor modifications as described(Casley et al. 2007). Allantoic fluids directly from eggs inoculatedwith viruses were fractionated on sodium dodecyl sulfate (SDS)-1 0%polyacrylamide gel, followed by transferring the samples to anitrocellulose filter. The nitrocellulose filter was then blocked with5% BSA/PBS at 37° C. for 1 hr. Following incubation of filters for 1 hrat 37° C. with rabbit antisera against HA peptides as described above,peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G (Sigma,Oakville, Canada) was added for an additional incubation of 1 h at roomtemperature, followed by chemiluminescent detection (ECL, AmershamPharmacia Biotech, Piscataway, N.J.). In some cases, dot blotting wasused to determine antigen-antibody interaction. The procedure isessentially the same as Western Blot except that the antigens (10 μl)were directly spotted on the nitrocellulose filter.

Quantitative detection of NA using antibodies against NA. The NAantigens were serially diluted in PBS-containing 0.01% Zwittergent(final concentration) and blotted onto PVDF membrane. The membrane isthen incubated with the universal antibodies HCA-2 Monoclonalantibodies, followed by detection with anti-rabbit IgG peroxidaseconjugate. Slot blot was conducted as described a similar procedure (LiC. et al. 2010). These were then scanned with a densitometer toquantitate. Standard curve for the quantification of NA by HCA-2. Thecurrently accepted 4-parameter logistic (4-PL) model was employed forthe calibration curve fitting in the immunoassays as described (Chun etal 2008) showing that there is a relationship between the amount ofsignal and the amount of protein. Therefore, antibodies to HCA-2 can beused as a quantitative detection method for detection of NA

Results: Selection of peptides for the generation of antibodies againstdiverse strains of influenza viruses Following a comprehensive analysisof the public database, the most conserved sequences were determined tobe the amino terminus of the NA and amino acids 221-231 (N2 numbering),largely agreeing with other investigators (Jackson et al. 1991; Horvathet al. 1998. Bianchi et al. 2005; Gerhard et al. 2006). However, it isof note that there are some minor variations as a result ofbioinformatics analyses (see Materials and Methods above). Both peptideswere relatively hydrophobic, thus presenting daunting challenge to thesubsequent peptide manipulations such as synthesis, purification,conjugation and epitope mapping in immunoassays.

The peptides, HCA-2 (SEQ ID NO: 1) and HCA-3 (SEQ OD NO: 2), were usedfor peptide modification and conjugation. Towards this end, the peptideswere first linked with a spacer (6-aminocaproic acid) to improveimmunogenicity (Scott et al. 1984), followed by a tripeptide (KKC) toenable solubilization of the peptide and also provide the cysteineresidue necessary for conjugation to the KLH carrier. The peptides orpeptide-conjugates were injected into NZW rabbits, followed by boostingevery two weeks. Significant antibody response was generated in NZWrabbits against both peptide conjugates. The antibodies generated wereIgG antibodies.

Binding specificities of the antibodies to diverse strains of theinfluenza viruses Experiments were then designed to determine whetherthe antibodies could bind to diverse strains of influenza strains. Tothis end, 9 subtypes of influenza viruses available at our institutionswere propagated in embryonated eggs. Allantoic fluid samples were useddirectly without any purification step so that the specificity ofantibodies could be determined at the same time. As shown in FIG. 6, theantibodies were found to bind all 9 subtypes. It is of note that thedifferences of reaction intensities amongst the subtypes were mostlylikely due to the different titres of virus in the crude allantoicfluids since the binding intensities with anti-HCA-2 and anti-HCA-3antibodies were largely consistent with that obtained with otheranti-influenza proteins (NP). Moreover, the different mobilities of theNA proteins amongst the influenza subtypes are due to the differences ofprotein size or processing stages of the NA proteins. Importantly, theantibodies demonstrated remarkable specificity against the influenza NAsas no binding of the antibodies to proteins derived from allantoicfluids were detected. As expected, the antibodies could also detect NAproteins from influenza virus grown in embryonated eggs and purified forthe preparation of human vaccines including influenza B (FIG. 3B).

While the preferred embodiments of the invention have been describedabove, it will be recognized and understood that various modificationsmay be made therein, and the appended claims are intended to cover allsuch modifications which may fall within the spirit and scope of theinvention.

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TABLE 1 Sequences SEQ ID NO: 1 HCA-2 .NILRTQESEC SEQ ID NO: 2 HCA-3MNPNQKIITIGS SEQ ID NO: 3 na NILRTQESSC

TABLE 2 Shannon entropy values for conserved region AA 221-232 of NA (N2numbering) AA # AA # AA # AA # AA # AA # AA # AA # AA # AA # 221 N 458 D138 Q 55 K 45 R 14 E 11 S 2 222 I 723 223 L 698 M 25 224 R 723 225 T 723226 Q 722 E 1 227 E 723 228 S 720 F 2 P 1 229 E 629 S 94 230 C 723 231 V428 A 155 Q 71 T 67 S 1 I 1 232 C 723 233 I 378 V 141 H 114 M 56 Q 24 L8 T 1 Y 1 234 N 498 D 80 K 68 Q 48 S 10 G 10 Y 4 R 3 E 1 H 1

TABLE 3 Shannon entropy values for conserved region AA 1-12 of NA AA #AA # AA # AA # AA # AA # AA # AA # AA # 1 M 639 — 84 2 N 644 — 76 D 1 K1 G 1 3 P 639 — 73 T 10 A 1 4 N 654 — 66 Q 1 T 1 I 1 5 Q 655 — 60 K 5 H1 P 1 L 1 6 K 654 — 49 R 19 E 1 7 I 646 — 44 L 33 8 I 614 — 35 F 32 L 32T 7 M 3 9 T 542 C 79 A 66 — 28 I 7 N 1 10 I 623 L 33 T 32 — 24 S 6 V 2 N1 Y 1 M 1 11 G 590 S 119 — 12 W 1 P 1 12 S 517 A 85 V 64 G 32 T 11 — 9 F4 I 1 13 V 386 I 218 T 83 A 27 — 6 L 3

The invention claimed is:
 1. A composition comprising an influenzaneuramidase peptide consisting of the amino acid sequence as set forthin SEQ ID No. 1 or SEQ ID No. 2 attached to the first end of a spacer, atripeptide linker KKC connected to the second end of the spacer and acarrier protein connected to the tripeptide linker.
 2. The compositionaccording to claim 1 wherein the spacer is selected from the groupconsisting of amino acids, peptides, phosphoramidite, ε-aminohexanoicacid and 6-aminocaproic acid.
 3. The composition according to claim 1wherein the carrier protein is selected from the group consisting ofkeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), rabbitserum albumin (RSA), ovalbumin (OVA), thyroglobulin (THY) and humangamma globulin (HGG).
 4. The composition according to claim 1 whereinthe spacer is 6-aminocaproic acid.
 5. A composition consisting of aninfluenza neuramidase peptide consisting of the amino acid sequence asset forth in SEQ ID No. 1 or SEQ ID No. 2 attached to the first end of aspacer, a tripeptide linker KKC connected to the second end of thespacer, and a carrier protein connected to the tripeptide linker.
 6. Thecomposition according to claim 5 wherein the carrier protein is selectedfrom the group consisting of keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA),thyroglobulin (THY) and human gamma globulin (HGG).
 7. The compositionaccording to claim 5 wherein the spacer is 6-aminocaproic acid.